Proper T-cell activation and effector function are essential for effective immunity. T-cell antigen receptor (TCR) signals are known to regulate the outcome of differentiation, but the mechanisms remain unclear. Recent work from our lab demonstrates that the Src family protein tyrosine kinase, p56Lck, is able to specifically link TCR signals to activation of the Mitogen Activated Protein Kinase (MAPK) pathway through the function of its SH3 domain. The MAPK pathway is known to be involved in T-cell activation downstream of TCR ligation and has previously been implicated in T-helper type 2 (Th2) effector function. We have utilized an Lck SH3 mutant knock-in mouse line (Lck W97A) to investigate the potential role of this regulatory signaling mechanism in determining T-lymphocyte activation and effector function. Our results demonstrate that the Lck SH3 domain function is required for normal activation of T-lymphocytes following TCR stimulation as indicated by significantly reduced proliferation, IL-2 production, and CD69 induction in Lck W97A T-cells. Biochemical studies confirm that activation of the MAPK pathway is selectively altered in Lck W97A T-cells as P-ERK1/2 induction is significantly reduced but phospho-PLCg1 induction and calcium mobilization is unaffected. In vivo experiments demonstrate a specific and significantly impaired Th2 immunity in Lck W97A mice, with reduced serum levels of IgG1, IgE and IL-4 following immunization with DNP-KLH, or infection with the helminth Nippostrongylus brasiliensis. Th1 immunity does not appear differentially regulated in Lck W97A mice as serum levels of IgG3 and IgG2b are similar to WT following immunization with DNP-KLH, as well as serum levels of IFN-g1 following immunization with heat-killed Brucella abortus. In vitro differentiation studies confirm that Lck W97A T-lymphocytes are able to be directed to the Th2 phenotype, as indicated by intracellular staining for IL-4, with significantly increased levels of IFN-g under Th2 differentiating conditions compared to WT. These data indicate that the Lck SH3 domain regulates activation of T-lymphocytes by affecting MAPK pathway induction and demonstrate a novel and critical role for Lck in the regulation of Th2-type immunity. The Lck SH3 domain has also been implicated in the pathogenesis of Plasmodium, the causative agent of malaria. The role of the mosquito vector on malaria pathogenesis is not well understood. Initial studies examining the role of vector salivary gland proteins on cells of the innate immune system indicate that Anopheles stephensi saliva is able to enhance macrophage activation and phagocytosis as well as enhance macrophage Ag-presentation to T-lymphocytes in an in vitro model.

Infection with the helminth parasite Schistosoma mansoni leads to chronic inflammation and Th2 mediated fibrosis, which result in severe pathology characterised by hepatosplenomegaly. Dendritic cells (DCs) are adept initiators of CD4+ T cell responses, but their fundamental importance in this regard in Th2 settings remains to be demonstrated. Indeed, the role of DCs at different stages of infection with S. mansoni is also yet to be determined. In addition, the importance of the interaction of DCs with tissue factors in the tissue microenvironment on the development of Th2 response to S. mansoni antigens is an area of active research and debate. This thesis is comprises of four studies. The first study tackles the involvement and importance of DCs in the induction and development of Th2 responses against S. mansoni using CD11c–diphtheria toxin receptor mice to deplete CD11c+ cells during the priming stage of the CD4+ Th2 response against S. mansoni. Diphtheria toxin treatment significantly depleted CD11c+ DCs from all tissues tested, with 70-80% efficacy. Even this incomplete depletion resulted in dramatically impaired CD4+ T cell production of Th2 cytokines, altering the balance of the immune response and causing a shift towards IFN-γ production. In contrast, basophil depletion using Mar-1 antibody had no measurable effect on Th2 induction in this system. These data underline the vital role that CD11c+ antigen presenting cells can play in orchestrating Th2 development against helminth infection in vivo, a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines. The second study addresses whether the exposure of DCs to the cercarial stage of the parasite is critical for either parasite survival or the subsequent development of the Th2 immune response against later stages of infection. It was found that CD11c depletion prior to infection resulted in increased parasite survival, but did not impair the development of CD4+ T cell Th2 response later in infection. The third study asked whether DCs continue to be necessary for the maintenance of the chronic immune response during infection with S. mansoni. In contrast, depletion of CD11c+ cells during the initiation (4 to 6 weeks) or maintenance (6 to 8 weeks or 12 to 14 weeks) of Th2 response to eggs, resulted in severely impaired Th2 cytokine production. Interestingly, depletion during the later stages of infection led to dramatic weight loss and mortality, coincident with impaired CD4+ T cell responses. These data suggest that CD11c+ antigen presenting cells, in addition to being important in the early priming phase, also play a vital role in the maintenance and homeostasis of chronic CD4+ T cell responses in a Th2 infection setting, the disruption of which can have lethal consequences. The final study in this thesis aimed to establish whether the tissue factor thymic stromal lymphopoietin (TSLP) is able to enhance or modulate the Th2 responses initiated by DCs stimulated with SEA. Contrary to previous studies, it was found that BMDCs do not become phenotypically activated by TSLP, in particular, they do not up-regulate the costimulatory molecule OX40L, nor does TSLP suppress the production of IL-12p40 or IL-12p70 in response to LPS or CpG. Further, exposure to TSLP had no impact on DC cytokine production or survival. Irrespective of this unaltered profile in vitro, TSLP exposed DCs transferred in vivo induced the production of significantly more Th1 and Th2 cytokines from polyclonally restimulated splenocytes than DCs exposed to medium alone. In addition to this, TSLP altered the kinetic of the immune response induced by DCs stimulated with the soluble egg antigen (SEA) of S. mansoni. This was characterised by the antigen specific production of T cell cytokines starting more rapidly than with non-TSLP treated control DCs. The alteration in the kinetics of the immune response was not restricted to Th2 antigens and was also seen to some extent in Propionibacterium acnes stimulated DCs. This suggests a possible role for TSLP in either inducing faster DC migration or greater production of T cell chemoattractants and thus, enhancing the rate of DC interaction with T cells.

To examine how dietary n-3 polyunsaturated fatty acids affect Th2 cell development, female C57BL/6 mice were fed a washout corn oil (CO) diet for 1 wk followed by 2 wk of either the same CO diet or a fish oil (FO) diet. CD4+ T cells were isolated from spleens and cultured under both neutral (anti-CD3 and phorbol myristate acetate (PMA)) and Th2 polarizing conditions (anti-CD3 and PMA, in presence of rIL-4, rIL-2, and anti-IFN-γ) in the presence of homologous mouse serum (HMS) or fetal bovine serum (FBS) for 2 d. Dietary n-3 PUFA significantly enhanced Th2 cell development and suppressed Th1 development under neutral conditions as assessed by intracellular cytokine staining for IL-4 and IFN-γ as the two prototypic Th2 and Th1 cytokines, respectively. However, under Th2 polarizing conditions, while the suppression of Th1 cells was maintained in FO-fed mice, no dietary effect was observed in Th2 cells. Dietary FO increased the Th2/Th1 ratio under both neutral and Th2 polarizing conditions with HMS in the cultures. To examine the effect of dietary n-3 PUFA on Th1 development, DO11.10 Rag2-/- mice expressing transgenic T cell receptor specific for ovalbumin (OVA) peptide were used. CD4+ T cells were isolated from spleens and lymph nodes and stimulated with ovalbumin (OVA) peptide and irradiated BALB/c splenocytes in the presence of rIL-12, anti-IL-4, and rIL-2 in HMS for 2d. Cells were expanded for another 3 d in the presence of rIL-2 and rIL-12. Dietary n-3 PUFA did not affect Th1 differentiation as assessed by the proportion of IFN-γ+, IL-4- T cells in the cultures, but suppressed rIL-2 induced expansion. The suppressed expansion was due to suppressed proliferation (p<0.05). In vivo expansion of antigen-specific T cells was visualized by flow cytometric analysis of CFSE-positive transgenic T cells. Dietary n-3 PUFA did not appear to affect antigen-induced CD4+ T cell cycle progression in vivo. Overall, these results suggest dietary n-3 PUFA have no direct effect on Th2 cell development but do directly suppress Th1 cell development following both mitogenic and antigenic stimulation in vitro.

Normal pregnancy and allergy are both characterized by a T helper (Th) 2 deviation. In the current study, we hypothesized that paternal antigen-induced cytokine responses during pregnancy would be deviated toward Th2 and an anti-inflammatory profile, and that the Th2 deviation would be more pronounced in allergic pregnant women. Blood samples were collected longitudinally on three occasions during pregnancy and two occasions post partum (pp). Of the 86 women initially included, 54 women had a normal pregnancy and completed the sampling procedures. Twelve women fulfilled the criteria for allergy (allergic symptoms and circulating immunoglobulin [Ig] E antibodies to inhalant allergens) and 20 were non-allergic (nonsensitized without symptoms). The levels of Th1- and Th2-associated cytokines and chemokines, the Th17 cytokine IL-17 and the anti-inflammatory cytokine IL-10 of the groups were compared. Paternal antigen-induced IL-4 and IL-10 responses increased between the first and the third trimester. Allergy was associated with decreased paternal antigen-induced IFN-γ and CXCL10 secretion in the nonpregnant state (one year pp) and also decreased IFN-γ/IL-4 and IFN-γ/IL-13 ratios during pregnancy. We also observed a decreased paternal antigen-induced IL-10 response in allergic compared with non-allergic women during pregnancy, along with a decreased IL-10/IL-13 ratio. In conclusion, our findings support the hypothesis of lower Th1 responses toward paternal antigens in allergic than in non-allergic women, but also indicate that allergy is associated with a lower capacity to induce anti-inflammatory IL-10 responses after paternal antigen stimulation during pregnancy. / <p>Funding Agencies|Swedish Research Council||Cancer and Allergy Association||Olle Engkvist Foundation||Vardal Foundation for Health Care Sciences and Allergy Research||National Swedish Association against Allergic Diseases||Linkoping University Hospital||</p>

INTRODUCTION: Allergic women have been reported to give birth to more children than non-allergic women, speculatively explained by the former's predisposition for Th2 polarization, possibly favoring pregnancy. AIM: The aim of this study was to test the hypothesis that allergy is associated with more Th2-deviated responses to paternal antigens throughout pregnancy. METHODS: Blood samples were collected on six occasions during pregnancy and two occasions postpartum (pp). Of the 86 women initially included, 54 women had a normal pregnancy and completed the sampling procedures. Eleven women fulfilled the strict criteria for allergy (allergic symptoms and circulating IgE antibodies to inhalant allergens) and 23 were strictly non-allergic (non-sensitized without symptoms). The numbers of blood mononuclear cells secreting IFN-gamma and IL-4, spontaneously and in response to paternal alloantigens, were compared between the groups. RESULTS: The numbers of spontaneously as well as paternal antigen-induced IFN-gamma- and IL-4-secreting cells were similar in allergic and non-allergic pregnant women on all occasions. A similar increase in the numbers of both IFN-gamma- and IL-4-secreting cells were found in allergic and non-allergic women during pregnancy, both regarding spontaneous and paternal antigen-induced secretion. CONCLUSIONS: This study does not support the hypothesis of a more pronounced Th2-deviation to paternal antigens in allergic pregnant women compared with non-allergic pregnant women, as measured by number of cytokine-secreting cells. The observed increase of both IFN-gamma- and IL-4-secreting cells during normal pregnancy may be interpreted as a Th2-situation, since the effects of IL-4 predominate over the effects of IFN-gamma.

The outcome of an infection is often determined by the qualitative nature of the immune response generated against the infectious agent. Various intracellular pathogens, including those that cause leprosy, tuberculosis, leishmaniasis, and most probably malaria and AIDS appear to require a predominant cell-mediated, Th1, response for effective containment, whereas the generation of a mixed Th1/Th2 or predominantly Th2 response is associated with progressive disease. Therefore, any attempt to develop universally efficacious vaccination against these pathogens must generate an immunological imprint that ensures a strong and stable cell-mediated response upon natural infection with the relevant pathogen. We report here critical tests of a strategy designed to achieve such an imprint using Bacille-Calmette-Guérin (BCG) vaccine. BCG vaccine is an attenuated form of M. bovis, the causative agent of tuberculosis in cattle, and is the most widely used vaccine in humans. However, considerable uncertainty still surrounds its efficacy against tuberculosis both in man and animals. As the protective dose is not known, BCG has been given at the maximum tolerable dose. However, recent studies in mice and other animals have shown that the dose of an antigen can be a critical factor in determining the type of immune response generated. I tested the general hypothesis that low dose vaccination would preferentially induce cell-mediated immune response and generate a Th1 imprint that can protect the host against intracellular pathogens in the particular case of mycobacteria. To this end, both adult and newborn mice were vaccinated with different doses of BCG or saline and cell-mediated and humoral immune responses were assessed at different times post-vaccination. Several weeks after vaccination, mice from each group were challenged with a dose of BCG that induces a mixed Th1/Th2 response in naïve mice, and the T-cell and antibody responses were assessed using ELISPOT and ELISA assays, respectively. The splenic bacterial burden was also determined using colony formation on agar plates. <p>Our results show that the class of immunity induced by BCG depends on the dose employed for vaccination, independent of the route of administration and the age and strain of mice used. In all cases, lower doses induce an exclusive cell-mediated, Th1, response with no antibody production, while higher doses induce either a mixed Th1/Th2 response or a predominantly Th2, humoral response, with higher titers of both IgG1 and IgG2a antibodies. Following intravenous high dose BCG challenge, all mice in the vaccinated groups developed a Th1 response associated with a more efficient clearance of BCG from the spleen. The greatest clearance of mycobacteria was generated following vaccination with lower doses, as low as 33 cfu of BCG. In addition, our findings demonstrate that newborn mice are not inherently biased towards generating Th2 responses, but they can generate Th1 responses and Th1 imprints if appropriate vaccination protocols (dose, route and time) are employed. Furthermore, subcutaneous exposure of young mice to environmental mycobacteria can induce a mixed Th1/Th2 response that can abrogate the potential to generate Th1 responses and Th1 imprints upon vaccination with low doses of BCG vaccine. Low dose neonatal BCG vaccination can circumvent the interference caused by impingement of environmental mycobacteria on the immune system. Therefore, our observations strongly support a neonatal low dose BCG vaccination strategy to provide universally efficacious protection against infections by pathogenic mycobacteria.

The outcome of an infection is often determined by the qualitative nature of the immune response generated against the infectious agent. Various intracellular pathogens, including those that cause leprosy, tuberculosis, leishmaniasis, and most probably malaria and AIDS appear to require a predominant cell-mediated, Th1, response for effective containment, whereas the generation of a mixed Th1/Th2 or predominantly Th2 response is associated with progressive disease. Therefore, any attempt to develop universally efficacious vaccination against these pathogens must generate an immunological imprint that ensures a strong and stable cell-mediated response upon natural infection with the relevant pathogen. We report here critical tests of a strategy designed to achieve such an imprint using Bacille-Calmette-Guérin (BCG) vaccine. BCG vaccine is an attenuated form of M. bovis, the causative agent of tuberculosis in cattle, and is the most widely used vaccine in humans. However, considerable uncertainty still surrounds its efficacy against tuberculosis both in man and animals. As the protective dose is not known, BCG has been given at the maximum tolerable dose. However, recent studies in mice and other animals have shown that the dose of an antigen can be a critical factor in determining the type of immune response generated. I tested the general hypothesis that low dose vaccination would preferentially induce cell-mediated immune response and generate a Th1 imprint that can protect the host against intracellular pathogens in the particular case of mycobacteria. To this end, both adult and newborn mice were vaccinated with different doses of BCG or saline and cell-mediated and humoral immune responses were assessed at different times post-vaccination. Several weeks after vaccination, mice from each group were challenged with a dose of BCG that induces a mixed Th1/Th2 response in naïve mice, and the T-cell and antibody responses were assessed using ELISPOT and ELISA assays, respectively. The splenic bacterial burden was also determined using colony formation on agar plates. <p>Our results show that the class of immunity induced by BCG depends on the dose employed for vaccination, independent of the route of administration and the age and strain of mice used. In all cases, lower doses induce an exclusive cell-mediated, Th1, response with no antibody production, while higher doses induce either a mixed Th1/Th2 response or a predominantly Th2, humoral response, with higher titers of both IgG1 and IgG2a antibodies. Following intravenous high dose BCG challenge, all mice in the vaccinated groups developed a Th1 response associated with a more efficient clearance of BCG from the spleen. The greatest clearance of mycobacteria was generated following vaccination with lower doses, as low as 33 cfu of BCG. In addition, our findings demonstrate that newborn mice are not inherently biased towards generating Th2 responses, but they can generate Th1 responses and Th1 imprints if appropriate vaccination protocols (dose, route and time) are employed. Furthermore, subcutaneous exposure of young mice to environmental mycobacteria can induce a mixed Th1/Th2 response that can abrogate the potential to generate Th1 responses and Th1 imprints upon vaccination with low doses of BCG vaccine. Low dose neonatal BCG vaccination can circumvent the interference caused by impingement of environmental mycobacteria on the immune system. Therefore, our observations strongly support a neonatal low dose BCG vaccination strategy to provide universally efficacious protection against infections by pathogenic mycobacteria.

To examine how dietary n-3 polyunsaturated fatty acids affect Th2 cell development, female C57BL/6 mice were fed a washout corn oil (CO) diet for 1 wk followed by 2 wk of either the same CO diet or a fish oil (FO) diet. CD4+ T cells were isolated from spleens and cultured under both neutral (anti-CD3 and phorbol myristate acetate (PMA)) and Th2 polarizing conditions (anti-CD3 and PMA, in presence of rIL-4, rIL-2, and anti-IFN-γ) in the presence of homologous mouse serum (HMS) or fetal bovine serum (FBS) for 2 d. Dietary n-3 PUFA significantly enhanced Th2 cell development and suppressed Th1 development under neutral conditions as assessed by intracellular cytokine staining for IL-4 and IFN-γ as the two prototypic Th2 and Th1 cytokines, respectively. However, under Th2 polarizing conditions, while the suppression of Th1 cells was maintained in FO-fed mice, no dietary effect was observed in Th2 cells. Dietary FO increased the Th2/Th1 ratio under both neutral and Th2 polarizing conditions with HMS in the cultures. To examine the effect of dietary n-3 PUFA on Th1 development, DO11.10 Rag2-/- mice expressing transgenic T cell receptor specific for ovalbumin (OVA) peptide were used. CD4+ T cells were isolated from spleens and lymph nodes and stimulated with ovalbumin (OVA) peptide and irradiated BALB/c splenocytes in the presence of rIL-12, anti-IL-4, and rIL-2 in HMS for 2d. Cells were expanded for another 3 d in the presence of rIL-2 and rIL-12. Dietary n-3 PUFA did not affect Th1 differentiation as assessed by the proportion of IFN-γ+, IL-4- T cells in the cultures, but suppressed rIL-2 induced expansion. The suppressed expansion was due to suppressed proliferation (p<0.05). In vivo expansion of antigen-specific T cells was visualized by flow cytometric analysis of CFSE-positive transgenic T cells. Dietary n-3 PUFA did not appear to affect antigen-induced CD4+ T cell cycle progression in vivo. Overall, these results suggest dietary n-3 PUFA have no direct effect on Th2 cell development but do directly suppress Th1 cell development following both mitogenic and antigenic stimulation in vitro.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.